Chymotrypsin from which the hydroxyl of Ser-195 has been eliminated (anhydrochymotrypsin) and which binds lima bean trypsin inhibitor (LBI) has been prepared. The LBI-binding anhydrochymotrypsin preparations are supposed to be catalytically inactive, but we find that they contain approximately 50 percent of a component which exhibits substantial reactivity towards p-nitrophenyl 3-phenylpropionate. Kinetics and inhibition tests show this component is not the native enzyme. We will attempt to separate the active and inactive components of LBI-binding anhydrochymotrypsin for further study. Affinity chromatogenphy on Sepharose linked ligands which include soybean trypsin inhibitor, turkey ovomucoid, epsilon-aminocaproyl-D-tryptophan methyl ester, and D-tryptophan methyl ester will be used in attempts to effect the separation. If the separation is successful, the active and inactive components will be characterized using a variety of physical and chemical techniques. Studies to learn into which of many possible conformations specific substrates of chymotrypsin and other serine proteases are locked in order to achieve good fit and high breakdown rates at the active sites of the enzymes will be continued. The effect of leaving group substituents and solvent deuterium on the rate of chymotryptic hydrolysis of a series of phenyl esters of S-N-carbomethoxy-2-amino-1, 2, 3, 4-tetrahydro-2-naphtoic acid will be determined to learn whether this geometrically constrained behaves as a specific or nonspecific substrate. Specific substrate phenyl esters are insensitive to leaving group electronic effects and exhibit KH/KD ratios of 2 to 3. Nonspecific substrates are leaving group sensitive and usually exhibit KH/KD ratios of less than 2. BIBLIOGRAPHIC REFERENCE: Michael S. Matta, Carmine M. Greene, Ross L. Stein, and Patricia A. Henderson, "Acylation of Subtilisin Carlsberg by Phenyl Esters", (1976) J. Biol. Chem., 251, 1006-1008.